First Report of Colletotrichum nymphaeae Causing Apple Bitter Rot in Southern Brazil.

Abstract

Bitter rot (BR) is a common disease on apples (Malus domestica Borkh.) worldwide. Fruit infection often occurs in warmer weather (25 to 32°C) where high temperatures and humidity strongly favor disease development. Three species causing BR have been reported: Colletotrichum gloeosporioides (Penz.) Penz. & Sacc. and its sexual stage Glomerella cingulata (Stoneman) Spaulding & Schrenk, C. acutatum J. H. Simmonds, and recently C. fragariae in Uruguay (1). In February 2012, typical BR symptoms were observed on apple fruits (cv. Gala) in two orchards located in Santa Catarina, Brazil. Initial symptoms were light brown, slightly sunken lesions that enlarged over time, becoming dark brown and forming sunken or saucer-shaped depressions that extended toward the fruit core in a V-shaped pattern. Acervuli were sparse and the conidia masses varied from dark gray to pale orange in color. To attempt to identify the causal organism, isolations were made from lesions on fully ripened fruits. Pure cultures were obtained following monosporic isolation and grown on PDA at 25°C with a 12-h photoperiod under fluorescent light for 7 to 15 days. The color of the upper surface of the colony varied from white to gray and was orange on the underside. One hundred conidia for each isolate were measured and each was one-celled, hyaline, fusiform, and with a length and width ranging between 8.0 and 25.9 μm (avg. 14.7), and between 2.4 and 9.9 μm (avg. 5.6), respectively. After germination, conidia formed oval appressoria between 5.1 and 9.9 μm (avg. 7.3) × 3.4 and 7.8 μm (avg. 5.2). These morphological characters are consistent with the description of C. nymphaeae (Pass.) Aa (2). To examine pathogenicity, eight 'Pink Lady' apples were inoculated with isolates MANE 25, 137, 143, and 144. A isolate treatments were arranged in a completely randomized design with three replications. Fruits were inoculated with two drops of 10 μl (1 × 106 conidia per ml) using wounded and non-wounded sterile fruit surfaces. Sterile distilled water drops served as controls. Fruit were incubated in a moist chamber at 25°C with a 12-h photoperiod for 10 days. Symptoms were observed at the inoculation site 3 to 4 days after inoculation (DAI) on wounded and 5 to 6 DAI on non-wounded fruits. All of the isolates produced symptoms identical to those observed in Santa Catarina, and each isolate was re-isolated from the apple lesions, confirming Koch's postulates. Fungal isolates were also characterized by sequencing of the internal transcribed spacer (ITS) rDNA using ITS1/IT4 primers and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) using GDF/GDR primers. Isolate sequences had 100% homology to C. nymphaeae. The nucleotide sequences were deposited in GenBank (KC840351, KC840352, KC840353, KC840354, KC875404, KC875405, KC875406, and KC875407). Species identified as C. nymphaeae are considered as part of the C. acutatum species complex and have been reported to occur on water lilies (Nymphaea alba) (3) and causing bitter rot on apples in Korea (2,4). To our knowledge, this is the first report of C. nymphaeae causing bitter rot of apples in southern Brazil.